Leishmania parasites cause a spectrum of devastating diseases in humans known as leishmaniasis. Current drug treatments are inadequate, often toxic, and no safe vaccine is available. A major obstacle in the development of new therapeutics is the lack of understanding on how Leishmania parasites establish infection in mammalian hosts. Previous studies on the surface glycoconjugates and sphingolipids in Leishmania have highlighted their importance in virulence. In contrast, functions of abundant membrane lipids such as phosphatidyl- ethanolamine (PE), phosphatidylcholine, and phosphatidylinositol remain extremely understudied in parasitic protozoans including Leishmania, although our recent data suggest their importance extends far beyond being basic membrane components. The long term goal is to elucidate the roles of membrane lipids and their metabolites in Leishmania infection. Interestingly, phospholipids in Leishmania parasites are drastically different from those in mammalian hosts. In particular, the vast majority of PE in Leishmania parasites belongs to plasmenylethanolamine (PLE), a group of phospholipids that is poorly understood. The overall objective of this application is to evaluate the contribution of PLE to the virulence of Leishmania parasites. The central hypothesis is that PLE biosynthesis is essential for the establishment of Leishmania infection. This hypothesis is formulated based on the following observations: 1) the abundance of PLE increases 2-4 fold as parasites differentiate from replicative, non-virulent procyclics to non-replicative, highly virulent metacyclics; 2) mutants defective in the production of EtN, which is required for the synthesis of PLE, showed greatly reduced level of PLE in stationary phase and failed to form infective metacyclics; and, 3) preliminary data indicate mutants defective in the degradation of sphingolipids, which could be the first step towards the synthesis of PLE, were severely attenuated in virulence. Together, these results suggest PLE biosynthesis is crucial for Leishmania infection. To test the central hypothesis, this proposal focuses on two candidate genes involved in the synthesis of PLE: LmEPCT and LmISC. Specific aims include: 1) to generate null mutants of LmEPCT and LmISC in L. major through targeted gene replacement; 2) to evaluate the impacts of LmEPCT and LmISC on Leishmania growth and infectivity; 3) to confirm the functions of LmEPCT and LmISC, and reveal the correlation between PLE biosynthesis and Leishmania virulence. Successful completion of the proposed studies will elucidate the contribution of PLE biosynthesis to Leishmania virulence; therefore complete an important step towards achieving the long term goal. Understanding the roles of phospholipids in Leishmania infection is highly significant, because it will not only provide fundamental insight into the mechanism of virulence, but also could reveal new therapeutic targets to help control these dangerous parasites. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: This proposal aims to elucidate the roles of a previously understudied group of phospholipids in the pathogenesis of Leishmania parasites, which cause a spectrum of devastating diseases in humans known as leishmaniasis. Successful completion of the proposed studies will provide new insight into the mechanism of virulence in Leishmania. Such findings will, ultimately, be used to improve and protect health. [unreadable] [unreadable] [unreadable]